Fox J C, Griffiths P D, Emery V C
University Division of Communicable Diseases, Royal Free Hospital School of Medicine, London, U.K.
J Gen Virol. 1992 Sep;73 ( Pt 9):2405-8. doi: 10.1099/0022-1317-73-9-2405.
The important goal of developing quantitative assays for viral nucleic acids in clinical samples has been achieved for human cytomegalovirus (HCMV) by using a modified polymerase chain reaction (PCR). A control PCR target sequence was constructed by PCR mutagenesis to allow the post-amplification quantification of HCMV DNA. The control region was identical to a naturally occurring sequence within the glycoprotein B (gB) coding part of the virus genome, except that a unique restriction site, introduced by the aforementioned mutagenesis step, allowed post-amplification differentiation of control/non-control target amplified product. This technique was initially validated using known amounts of cloned control/non-control target DNA, and was found to be sufficiently sensitive to allow the quantification of a range of 10 to 10(6) genome equivalents of virus. The method was applied to urine samples of congenitally infected infants for which infectious virus titres were available. The results obtained demonstrated that the number of infectious virions determined by conventional cell culture represented a small proportion of the HCMV genome present in the samples, as assessed by the quantitative PCR methodology.
通过使用改良的聚合酶链反应（PCR），已经实现了对临床样本中人类巨细胞病毒（HCMV）病毒核酸进行定量检测这一重要目标。通过PCR诱变构建了一个对照PCR靶序列，以实现HCMV DNA扩增后的定量分析。该对照区域与病毒基因组糖蛋白B（gB）编码部分内的一个天然存在的序列相同，只是上述诱变步骤引入的一个独特限制性位点使得对照/非对照靶标扩增产物在扩增后能够区分开来。该技术最初使用已知量的克隆对照/非对照靶标DNA进行验证，结果发现其灵敏度足以对10至10⁶个病毒基因组当量范围进行定量分析。该方法应用于可获得感染性病毒滴度的先天性感染婴儿的尿液样本。所获得的结果表明，通过定量PCR方法评估，传统细胞培养确定的感染性病毒粒子数量仅占样本中存在的HCMV基因组的一小部分。
